Hsp70, in Combination with IL-15 and PD-1 Blocker, Interferes with The Induction of Cytotoxic NK Cells in Relapsed Acute Myeloid Leukemia Patients

Natural killer (NK) cells are critical immune cells for acute myeloid leukemia (AML) targeting. However,little is known about the relationship between using checkpoint inhibitors and heat shock protein 70 (Hsp70) as NK cellactivators to control AML. Therefore, the study aims to find the best formulation of Hsp70, human PD-1 (Programmedcell death protein 1) blocker, and interleukin 15 (IL-15) to activate NK cells against AML. In this experimental study, the NK cells were isolated from mononuclear cells (MNCs) byusing magnetic activation cell sorting (MACS) and were activated using the different combinations of Hsp70, PD-1blocker, and IL-15 and then followed by immunophenotyping, functional assays to estimate their killing potential, andevaluation of expression pattern of PRF1, PIK3CB, PD-1, AKT-1, FAS-L, TRAIL, and GER A and B. The expression of PD-1 was significantly (P<0.05) reduced after NK cell activation by the different formulas ofIL-15, Hsp70, and PD-1 blocker. The expression of NKG2A in the treated NK cells was reduced particularly in the IL-15(P<0.01) and IL-15+PD-1 blocker (P<0.05) groups. The addition of Hsp70 increased its expression. The cytotoxic effectof NK cells increased in all groups, especially in IL-15+PD-1 blocker besides increasing interferon-gamma (IFN-γ),Granzymes, and perforin expression (P<0.05). All IL-15+PD-1 blocker group changes were associated with the upregulationof PIK3CB and AKT-1 as key factors of NK cell activation. The presence of Hsp70 reduced IFN-γ releasing,and down-regulation of PIK3CB, AKT-1, Granzymes, and Perforin (P<0.05). We suggested the combination of IL-15 and PD-1 blocker could enhance the killing potential of AMLNKcells. Moreover, Hsp70 in combination with IL-15 and PD-1 blocker interferes activation of AML-NK cells throughunknown mechanisms.